Exchange of endogenous and heterogeneous yeast terminators in <i>Pichia pastoris</i> to tune mRNA stability and gene expression

Ito, Yoichiro; Terai, Goro; Ishigami, Misa; Hashiba, Noriko; Nakamura, Yasuyuki; Bamba, Takahiro; Kumokita, Ryota; Hasunuma, Tomohisa; Asai, Kiyoshi; Ishii, Jun; Kondo, Akihiko

doi:10.1093/nar/gkaa1066

Cited by 40 publications

(36 citation statements)

References 41 publications

(40 reference statements)

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“…This strain can be used for maximized production of other isoprenoid products including squalene ( Paradise et al, 2008 ; Ding et al, 2014 ). Ito et al (2020) showed tuneable gene expression in P. pastoris using endogenous terminators of S. cerevisiae ( Ito et al, 2020 ). Here, we also report the successful transferability of terminators and promoters, such as the CYC1 minimal promoter, from S. cerevisiae to P. pastoris ( Figure 5A ), in addition to plant-derived ATF/BSs.…”

Section: Discussionmentioning

confidence: 99%

“…By employing only nine (three weak, three medium, and three strong) plant-derived ATFs and their corresponding BSs, we previously established a combinatorial library of S. cerevisiae variants that allow a wide range of gene expression fine-tuning ( Naseri et al, 2019 ). Considering the similarity between S. cerevisiae and P. pastoris , functional regulators of S. cerevisiae are expected to be transferable to P. pastoris ( Ito et al, 2020 ). A recent study showed that among the 72 terminators in S. cerevisiae , 41 had a high degree of transferability to P. pastoris ( Ito et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

“…Considering the similarity between S. cerevisiae and P. pastoris , functional regulators of S. cerevisiae are expected to be transferable to P. pastoris ( Ito et al, 2020 ). A recent study showed that among the 72 terminators in S. cerevisiae , 41 had a high degree of transferability to P. pastoris ( Ito et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris

Naseri

Prause

Hamdo

2021

Front. Bioeng. Biotechnol.

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The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has become a powerful eukaryotic expression platform for biopharmaceutical and biotechnological applications on both laboratory and industrial scales. Despite the fundamental role that artificial transcription factors (ATFs) play in the orthogonal control of gene expression in synthetic biology, a limited number of ATFs are available for P. pastoris. To establish orthogonal regulators for use in P. pastoris, we characterized ATFs derived from Arabidopsis TFs. The plant-derived ATFs contain the binding domain of TFs from the plant Arabidopsis thaliana, in combination with the activation domains of yeast GAL4 and plant EDLL and a synthetic promoter harboring the cognate cis-regulatory motifs. Chromosomally integrated ATFs and their binding sites (ATF/BSs) resulted in a wide spectrum of inducible transcriptional outputs in P. pastoris, ranging from as low as 1- to as high as ∼63-fold induction with only small growth defects. We demonstrated the application of ATF/BSs by generating P. pastoris cells that produce β-carotene. Notably, the productivity of β-carotene in P. pastoris was ∼4.8-fold higher than that in S. cerevisiae, reaching ∼59% of the β-carotene productivity obtained in a S. cerevisiae strain optimized for the production of the β–carotene precursor, farnesyl diphosphate, by rewiring the endogenous metabolic pathways using plant-derived ATF/BSs. Our data suggest that plant-derived regulators have a high degree of transferability from S. cerevisiae to P. pastoris. The plant-derived ATFs, together with their cognate binding sites, powerfully increase the repertoire of transcriptional regulatory modules for the tuning of protein expression levels required in metabolic engineering or synthetic biology in P. pastoris.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris

Naseri

Prause

Hamdo

2021

Front. Bioeng. Biotechnol.

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“…In addition, recent work has shown that terminator sequences can be designed as tunable control elements that can be “turned on” to attenuate gene transcription at low temperatures ( Roßmanith et al, 2018 ). With the growing evidence that the structural components of terminators may be malleable depending on species, future work should focus on understanding the combined contributions of terminator components, including those beyond transcriptional control (e.g., modulation of protein expression) for metabolic engineering ( Curran et al, 2013 ; Ito et al, 2020 ). This may lead to better designs for strong synthetic terminators with consistent cross-species performance.…”

Section: Discussionmentioning

confidence: 99%

Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species

Gale

McCormick

2021

Front. Microbiol.

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Cyanobacteria utilize sunlight to convert carbon dioxide into a wide variety of secondary metabolites and show great potential for green biotechnology applications. Although cyanobacterial synthetic biology is less mature than for other heterotrophic model organisms, there are now a range of molecular tools available to modulate and control gene expression. One area of gene regulation that still lags behind other model organisms is the modulation of gene transcription, particularly transcription termination. A vast number of intrinsic transcription terminators are now available in heterotrophs, but only a small number have been investigated in cyanobacteria. As artificial gene expression systems become larger and more complex, with short stretches of DNA harboring strong promoters and multiple gene expression cassettes, the need to stop transcription efficiently and insulate downstream regions from unwanted interference is becoming more important. In this study, we adapted a dual reporter tool for use with the CyanoGate MoClo Assembly system that can quantify and compare the efficiency of terminator sequences within and between different species. We characterized 34 intrinsic terminators in Escherichia coli, Synechocystis sp. PCC 6803, and Synechococcus elongatus UTEX 2973 and observed significant differences in termination efficiencies. However, we also identified five terminators with termination efficiencies of >96% in all three species, indicating that some terminators can behave consistently in both heterotrophic species and cyanobacteria.

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“…Ito et al characterized 72 terminators derived from P. pastoris , S. cerevisiae , and synthetic terminators, and found that the tunable range could reach up to 17-fold. Interestingly, the S. cerevisiae terminators could maintain their function after being transferred to P. pastoris [ 61 ]. These preliminary studies indicated the significance of terminators in regulating the expression level of heterologous genes and more mechanistic studies should be carried out in the near future.…”

Section: Synthetic Biology Toolkit For P Pastorismentioning

confidence: 99%

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

Gao

Jiang

Lian

2021

Synthetic and Systems Biotechnology

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The methylotrophic yeast Pichia pastoris (a.k.a. Komagataella phaffii ) is one of the most commonly used hosts for industrial production of recombinant proteins. As a non-conventional yeast, P. pastoris has unique biological characteristics and its expression system has been well developed. With the advances in synthetic biology, more efforts have been devoted to developing P. pastoris into a chassis for the production of various high-value compounds, such as natural products. This review begins with the introduction of synthetic biology tools for the engineering of P. pastoris , including vectors, promoters, and terminators for heterologous gene expression as well as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated System (CRISPR/Cas) for genome editing. This review is then followed by examples of the production of value-added natural products in metabolically engineered P. pastoris strains. Finally, challenges and outlooks in developing P. pastoris as a synthetic biology chassis are prospected.

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Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression

Cited by 40 publications

References 41 publications

Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris

Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris

Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

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